For the functional analysis of many genes, investigators need to isolate and manipulate large DNA fragments. The advent of genomics and the study of genomic regions of DNA have generated a need for vectors capable of carrying large DNA regions.
In general, two types of yeast vector systems are presently available. The first type of vector is one capable of transferring small insert DNA between yeast and bacteria. A second type of vector is a fragmenting vector which creates interstitial or terminal deletions in yeast artificial chromosomes (YACs). The small insert shuttle vectors are able to recombine with and recover homologous sequences. They are centromere-based and replicate stably and autonomously in yeast, but also contain a high-copy origin of replication for maintenance as bacterial plasmids. However, these vectors are limited by their small insert capacity. The second type of vector (also known as fragmenting vectors) has recombinogenic sequences, but is unable to transfer the recovered insert DNA to bacteria for large preparations of DNA.
Researchers use fragmentation techniques to narrow down the region of interest in YACs. However, isolating sufficient quantities of YAC DNA from agarose gels for microinjection or electroporation remains cumbersome. Purification remains a problem when the YAC comigrates with an endogenous chromosome. In addition, YACs may be chimeric or contain additional DNA regions that are not required for the particular functional study.
Types of vectors available for cloning large fragments in bacteria are cosmids, P1s and bacterial artificial chromosomes (BACs). These vectors are limited to bacteria and cannot be shuttled to yeast for modification by homologous recombination. Bacterial vectors are also limited in their use for transforming plants and algae. For example, though chloroplasts are thought to originate from the endosymbiosis of photosynthetic bacteria into eukaryotic hosts translation of chloroplasts in more complex. Adding to the complexity of genetically engineering plants and algae is the presence of multiple chloroplasts with multiple copies of the chloroplast genome. Thus, there exists a need for developing a method to express proteins from large fragments of DNA in the chloroplasts of plants and algae